Chrysene, one of polycyclic aromatic hydrocarbons, is discharged in the environment because of incomplete combustion. It has been used for organic material synthesis or chemical research, but has not been produced as a commercial product. In the environment, the movement of chrysene is hardly detected in soil because it does not have volatility and it is absorbed onto sediment and suspended solids in water, indicating it does not have volatility on the surface of water, either. Decomposition of a microorganism is slower in water than in soil. Absorption in the soil makes the decomposition of a microorganism even slower. Hydrolysis of it does not occur in the environment and the level of its concentration in aquatic living organisms is very low. Chrysene exists as particles in the air and thus it can be eliminated by physical methods.
According to a report by the Ministry of Environment Korea (1997) investigating chrysene concentration in the domestic air and underground shopping centers, the chrysene concentration was 1.80 ng/m3 in Seoul residential area and 1.2 ng/m3 in Taepyeong-dong, Daejeon, Korea. To investigate food contamination by polycyclic aromatic hydrocarbons, the chrysene concentrations in meat processed products sold in 9 cities in Korea were examined. As a result, the concentration of chrysene was 0.176 μg/kg in smoked products, 0.842 μg/kg in poultry, 0.143 μg/kg in meats (Kim M C et al., Korea Food & Drug Administration, 2001 Endocrine Discruptors Research report, 2001, and 0.82 μg/kg in edible oil and fat (Lee J O et al., Korea Food & Drug Administration, Research report 6, 790. 2002).
Chrysene was absorbed via oral pathway in animal tests. It was also absorbed through skin but the absorption rate was very slow and most of the absorbed chrysene was discharged through feces [ASTDR (Agency for Toxic Substance and Disease Registry), 1990). Chrysene is converted into dihydrodiols by an enzyme and further changed into diol-epoxide capable of binding to DNA to produce DNA byproduct. It was reported that diol-epoxide derivatives of chrysene could be potential carcinogens or mutagens. In Ames tests using salmonella TA110 and TA98 strains, chrysene showed mutagenicity in rats even under normal metabolic activity of the liver [HSDB (Hazardous Substance Data bank)].
Reports on the carcinogenicity of the polycyclic aromatic hydrocarbons and changes of gene expression induced by the same have been made intermittently but those reports are mostly limited to benzo[a]pyrene, the most representative polycyclic aromatic hydrocarbon. Despite chrysene has high potential for causing cancer in human, there are not enough data to evaluate risk in human and the screening method is still limited to the conventional GC-MS (Gas Chromatography-Mass Spectrometer) and HPLC (High Performance Liquid Chromatography). GC-MS or HPLC enables quantification but requires high cost equipments and set up for proper conditions for analysis. Therefore, a faster and easier screening method such as real-time PCR (real-time reverse transcript polymerase chain reaction) with primers or DNA microarray chip is required to establish a molecular index to detect toxicity and to evaluate risk in human. It is also important to establish a countermeasure for the exposure on chrysene and a regulation method of the same by using the above method.
Genome sequencing project of various species including 6 species of mammals and 292 species of microorganisms has been completed and reported to NCBI (National Center for Biotechnology Information). Based on this huge data, genome-wide expression study has been undergoing to disclose functions of those genes. DNA microarray is also performed to analyze expressions of thousands of genes at a time (Schena M et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996).
Microarray is prepared by integrating cDNA (complementary DNA) or 20-25 base pair long oligonucleotide sets on a glass substrate. cDNA microarray has been produced in laboratories and companies such as Agilent and Genomic Solutions, in which cDNAs are fixed by a mechanical method or ink jetting on a chip (Sellheyer K and Belbin T J, J. Am. Acad. Dermatol. 51: 681-692, 2004). Oligonucleotide microarray has been produced in Affymetrix by direct synthesis on a chip via photolithography and in Agilent by fixing synthetic oligonucleotides on a chip (Sellheyer K et al., Am. Acad. Dermatol. 51: 681-692, 2004).
To analyze gene expression, RNAs are extracted from samples such as tissues, which are hybridized with oligonucleotide on DNA microarray. The obtained RNA is labeled with a fluorescent material or an isotope and converted into cDNA. In oligo-microarray, the control and the experimental groups are labeled with two different fluorescent materials (for example, Cy3 and Cy5), followed by hybridization simultaneously on the chip. Images are optically scanned to measure the strength of fluorescence and the results are analyzed. By comparing the strengths of the two different fluorescent materials, gene expression is determined (Somasundaram K et al., Genomics Proteomics I: 1-10, 2002).
The recent DNA microarray based high tech toxicogenomics enables the analysis and quantification of gene expression patterns in specific tissues or cell lines induced by a medicant or novel a drug candidate and other chemicals. That is, by analyzing the expression frequency of a specific gene in a specific cell, it is possible to identify a gene causing side effects of such drugs or harmful action of such environmental pollutant materials. Then, it is further possible to understand the molecular mechanism related to such side effects and functions of drugs and environmental pollutant materials.
The present inventors screened genes up-regulated or down-regulated by chrysene by observing and analyzing gene expression profiles of chrysene in HepG2, a human Hepatoma cell line, using oligo-microarray on which 41,000 human genes are integrated, and further completed this invention by confirming the genes capable of confirming the exposure on chrysene by investigating expressions of target genes by real-time RT-PCR and by establishing a method for confirming the exposure on chrysene.